The present invention relates to the novel 1,4,7,10-tetraazacyclododecane-1,4-diacetic acid derivatives of formula (I), their complexes with paramagnetic metal ions and physiologically compatible salts thereof, as well as to the preparation thereof and the use thereof for the preparation of chelating agents. 
In particular the present invention relates to the derivatives of the novel compound, 1,4,7,10-tetraazacyclododecane-1,4-diacetic acid of formula (X), its chelated complex salts with pararnagnetic metal ions and the physiologically compatible salts thereof, as well as to the preparation thereof and the use thereof for the preparation of chelating agents. 
The compounds of general formula (I), and in particular the compound of formula (X), are novel chelating agents for bi- trivalent metal ions and are also important intermediates for the synthesis of 1,4,7,10-tetraazacyclododecane derivatives chelating agents, symmetrically functionalized at the 1- and 4-positions.
The compounds of general formula (I) are therefore the starting material for the synthesis of multidentate derivatives which are capable of complexing different metals, some of which have applications in the biomedical field, such as gadolinium complexes of said derivatives, which are used in diagnostic as contrast agents for the magnetic resonance technique (Magnetic Resonance Imaging, MRI).
In particular, the medical diagnosis by means of xe2x80x9cMagnetic Resonance Imagingxe2x80x9d (M.R.I.), which is known to be a powerful diagnostic means in the clinical practice (Stark, D. D., Bradley, W. G., Jr., Eds. xe2x80x9cMagnetic Resonance Imagingxe2x80x9d The C. V. Mosby Company, St. Louis, Mo. (USA), 1988), mainly makes use of paramagnetic pharmaceutical compositions, preferably containing chelated complex salts of bi- and trivalent paramagnetic metal ions with aminopolycarboxylic acids and/or derivatives or analogues thereof.
Presently some of them are used in clinic as M.R.I. contrast agents (Gd-DTPA, N-methylglucamine salt of the gadolinium complex with diethylenetriaminopentaacetic acid, MAGNEVIST(copyright), Schering; Gd-DOTA, N-methylglucamine salt of the gadolinium complex with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTAREM(copyright), Guerbet).
A list of significant patent literature for illustrating the prior art in this diagnostic field, although only exemplary and incomplete, is the following: EP 71564 (Schering), U.S. Pat. No. 4,639,365 (Sherry), U.S. Pat. No. 4,615,879 (Runge), DE-A-3401052 (Schering), EP 130934 (Schering), EP 65728 (Nycomed), EP 230893 (Bracco), U.S. Pat. No. 4,826,673 (Mallinckrodt), U.S. Pat. No. 4,639,365 (Sherry), EP 299795 (Nycomed), EP 258616 (Salutar), WO 8905802 (Bracco).
Object of the present invention are the compounds of general formula (I): 
in which
R is a hydrogen atom, a straight or branched or cyclic C1-C6 alkyl group, unsubstituted or substituted by 1 to 10 oxygen atoms, or a C1-C20 alkyl group, optionally interrupted by a phenylene, in its turn substituted by a straight or branched C1-C6 alkyl group, unsubstituted or substituted by 1 to 3 C1-C7 groups; the aromatic group being unsubstituted or substituted by alkoxy, carboxy, sulfamoyl, hydroxyalkyl, amino groups; as well as their complexes with bi-trivalent metal ions having atomic number variable from 20 to 31, 39, 42, 43, 44, 49, or from 57 to 83, as well as their salts with anions of physiologically acceptable organic acids, selected, for example, from acetate, succinate, citrate, fumarate, maleate, oxalate, or with anions of inorganic acids such as halo acids ions, namely chlorides, bromides, iodides. Metal ions suitable for preparing complex salts with the novel chelating agents of general formula (I) are mainly bivalent or trivalent ions of the elements having atomic number variable from 20 to 31, 39, 42, 43, 44, 49, or from 57 to 83; particularly preferred being Fe(2+), Fe(3+), Cu(2+), Cr(3+), Gd(3+), Eu(3+), Dy(3+), La(3+), Yb(3+) or Mn(2+) or also radioisotopes as 51Cr, 67Ga, 68Ga, 111In, 99mTc, 140La, 175Yb, 153Sm, 166Ho, 90Y, 149Pm, 177Lu, 47Sc, 142Pr, 159Gd, 212Bi.
Preferably R can be selected from the group consisting of: H or a straight or branched alkyl group, such as a methyl, ethyl, propyl, isopropyl, butyl, isobutyl group, in its turn substituted by hydroxy groups or interrupted by oxygen atoms, as defined above.
When an aromatic group is present in R, particularly preferred are the phenyl and benzyl groups.
Particularly preferred are the compounds of formula (I) selected from the group consisting of: xcex1,xcex1xe2x80x2-dimethyl-1,4,7,10-tetraazacyclododecane-1,4-diacetic acid; xcex1,xcex1xe2x80x2-diethyl-1,4,7,10-tetraazacyclododecane-1,4-diacetic acid; and xcex1,xcex1xe2x80x2-dibenzyl-1,4,7,10-tetraazacyclododecane-1,4-diacetic acid.
Preferred anions of inorganic acids suitable for salifying the chelated complexes of the invention comprise, in particular, halo acids ions such as chlorides, bromides, iodides or other ions, such as sulfate.
Preferred anions of organic acids suitable for the above aim comprise those of the acids conventionally used in pharmaceutical technique for the salification of basic substances, such as acetate, succinate, citrate, fumarate, maleate.
Preferred amino acids anions comprise, for example, those of taurine, glycine, lysine, arginine or ornithine, or of aspartic and glutamic acids.
The formation of the complex metal salt is carried out conventionally, preferably in water or in a suitable water-alcohol mixture, by reacting compounds (I) with a metal salt (oxide or halide), whereas the temperature can range from 25xc2x0 C. to 100xc2x0 C., preferably from 40xc2x0 C. to 80xc2x0 C.
The choice of the metal ion and of any neutralizing ion is closely related to the intended use of the complex to be prepared.
The preparation of the novel compound, manganese complex of 1,4,7,10-tetraazacyclododecane-1,4-diacetic acid, which is in the neutral form and therefore does not require the formation of a physiologically compatible salt, is described in the Experimental section. 
The novel compounds of the present invention have a good tolerability; moreover, their water solubility and the low osmolality of their solutions are a further advantageous characteristic which makes them particularly suitable for use in nuclear magnetic resonance.
Both soluble and less soluble compounds are useful for the oral and enteral administrations and, therefore, for the imaging of the GI tract.
As far as the parenteral administration is concerned, the compounds are preferably formulated as a sterile aqueous solution or suspension, whose pH can range for instance from 6.0 to 8.5.
These aqueous solutions or suspensions can be administered in concentrations ranging from 0.002 to 1.0 mol.
These formulations can be lyophilized and supplied as they are for reconstitution before use. For the GI use or for the injection in the body cavities, such agents can be formulated as a solution or suspension containing suitable additives, for example thickeners.
In the oral administration they can be formulated according to preparation methods commonly used in the pharmaceutical practice possibly as coated formulations in order to get additional protection from the stomach acid pH, by preventing the release of the chelated metal ion occurring in particular at pH typical of gastric juices.
Other excipients, for instance sweeteners and/or flavouring agents, can also be added according to known techniques of pharmaceutical formulation.
The chelated complex salts of this invention can also be used as radioharmaceuticals in nuclear medicine, both in the diagnostic and therapeutic sector.
In this case, however, the metal ion which is chelated is a radioisotope, for instance 51Cr, 67Ga, 68Ga, 111In, 99mTc, 140La, 175Yb, 153Sm, 166Ho, 90Y, 149Pm, 177Lu, 47Sc, 142Pr, 159Gd and 212Bi.
The compounds of the invention can optionally be chemically conjugated with suitable macromolecules or included in suitable carriers.
It is also an object of the invention the process for the preparation of the compounds of general formula (I) starting from triethylenetetramine of formula (II) comprising the following steps represented in Scheme 1: 
a) condensation of triethylenetetramine of formula (II) with a glyoxal derivative (glyoxal hydrate, or Bertagnini""s salt), in water or in water-soluble solvents or in mixtures thereof, at a temperature of 0-50xc2x0 C., in the presence of a stoichiometric amount or a slight excess of calcium hydroxide to give octahydro-3H,6H-2a,5,6,8a-tetraazacenaphthylene of formula (III);
b) condensation of the compound from step a) with an alkylating agent Xxe2x80x94CH2xe2x80x94CH2xe2x80x94X, in which X is Cl or Br in amounts from 1 to 5 mols per mol of compound (III), in a dipolar aprotic solvent and in the presence of a base selected from alkali or alkaline-earth metal carbonates, in amounts from 5 to 10 mols per mol of compound (III), and with the addition of NaY, wherein Y is I or Br, as catalyst in amounts from 0.1 to 2 mols per mol of compound (III), wherein X and Y are not at the same time Br, at a temperature from 25 to 150xc2x0 C., to give decahydro-2a,4a,6a,8a-tetraazacyclopent[fg]acenaphthylene of formula (IV);
c) oxidation of compound (IV) with bromine at pH 4-5 to give 2,3,4,6,7,8-hexahydro-1H,5H-4a,6a-diaza-2a,8a-azoniacyclopent[fg]acenaphthylene dibromide of formula (V);
d) formation of 2,3,4,6,7,8-hexahydro-1H,5H-4a,6a-diaza-2a,8a-azoniacyclopent[fg]-acenaphthylene diperchlorate of formula (VI), poorly water-soluble, by addition of perchlorate ions to the solution of compound (V);
e) alkylation of compound of formula (VI) with an alkylating agent of formula Rxe2x80x94CH(X)COOH, in which X is a halogen, and R is defined as above, in basic conditions, to give the novel compounds of formula (VII);
f) hydrolysis of the formyl group of compounds (VII) in basic conditions to give the novel compounds of formula (VIII);
g) alkylation of compound (VIII) with the alkylating agent of formula Rxe2x80x94CH(X)xe2x80x94COOH, in which X and R have the meanings defined above, in basic conditions, to give the novel compounds of formula (IX);
h) final hydrolysis of compounds (IX), in basic conditions, to give the compounds of formula (I).
The process of the present invention requires no isolation of the intermediate compounds, although this has been carried out to correctly identify their structures by means of various analytic techniques.
The process of the invention is particularly useful from the industrial point of view since it allows to prepare compounds (I) from inexpensive precursors such as triethylenetetramine, instead of expensive macrocyclic derivatives.
Steps a) and b) are object of Italian Patent application MI 97A000783, in the Applicant""s name, and provide compound (IV) in good yields.
In particular, in step b), the alkylating agent is generally added in amounts from 1 to 5 mols per mol of compound (III).
The reaction takes place in dipolar aprotic solvents, preferably selected from the group consisting of: DMAC (dimethylacetamide), DMF (dimethylformamide), DMSO (dimethylsulfoxide) and N-methyl-pyrrolidone; and in the presence of an inorganic base, preferably an alkali metal carbonate.
The temperature, depending on the solvent and on the alkylating agent, can range from 25 to 150xc2x0 C., preferably from 30 to 80xc2x0 C. The reaction time is 1-48 h.
More specifically, when using 1,2-dichloroethane and NaBr, temperature ranges from 50 to 80xc2x0 C. and the reaction time ranges from 2 to 5 h.
When using 1,2-dichloroethane and NaI, temperature ranges from 30 to 50xc2x0 C. and the reaction time ranges from 5 to 15 h.
Step c) is the oxidation with bromine in amounts of 2-2.5 mols per mol of compound (IV) in aqueous solution at pH of 4-5, obtained by addition of an acid and kept during the reaction by addition of a base, preferably NaOH or KOH.
The reaction temperature ranges from 17 to 30xc2x0 C. and the reaction time is usually 16 hours.
At the end of the reaction, and this is step d) of the present invention, sodium perchlorate or perchioric acid are added in amounts ranging from 2.5 to 3 mols per mol of compound (IV), thereby precipitating compound (VI) in good yields.
The alkylation conditions in step e) are usual in literature.
Particularly preferred are the alkylating agents of formula Rxe2x80x94CH(X)xe2x80x94COOH in which X is bromine or chlorine and most preferred are the alkylating agents of formula XCH2COOH, in which R is the hydrogen atom and X is bromine or chlorine.
The alkylation reaction is usually carried out in the following conditions: the reaction temperature can range from 30 to 70xc2x0 C.; the reaction time usually ranges from 10 to 25 hours; basic pH from 10 to 12 is obtained by addition of a base, preferably sodium or potassium hydroxide; the amount of alkylating agent is stoichiometric or in a slight excess (1÷2 mols).
Step f) is the hydrolysis of compounds (VII) in aqueous solution at pH made basic by addition of a base, preferably NaOH or KOH, at temperatures ranging from 50 to 100xc2x0 C.
Step g) is effected as already described for step e).
Step h) is the hydrolysis of compounds (IX) in aqueous solution at pH made basic by addition of 3-7 mols of a base, preferably NaOH or KOH, at temperatures ranging from 150-220xc2x0 C.
Particularly preferred is the process of the invention for the preparation of compound (X), according to the following Scheme 2: 
in which in step e) and g) the alkylating agent is the compound of formula XCH2COOH and through formation of the novel intermediate compounds:
7-formyl-1,4,7,10-tetraazabicyclo[8.2.1]tridecane-13-on-4-acetic acid of formula (VIIa); 1,4,7,10-tetraazabicyclo[8.2.1]tridecane-13-on-4-acetic acid of formula (VIIIa); 1,4,7,10-tetraazabicyclo[8.2.1]tridecane-13-on-4,7-diacetic acid of formula (IXa).
The preferred conditions in the presence of BrCH2COOH as alkylating agent, are at least 1.5 mols of alkylating agent per mol of starting product and the temperature is 45xc2x0 C.; the reaction time is 21 hours; and pH is 11.5.
Steps e) and 9) are carried out using the same alkylating agent, namely BrCH2COOH, and the same conditions can therefore be used for the two steps.
It is moreover possible to directly alkylate compound (V) or compound (VI) with at least 4 mols of XCH2COOH, preferably BrCH2COOH, per mol of starting product, at a temperature of 90xc2x0 C. and at basic pH, thus obtaining compound (IXa), without the step of formation of the intermediates shown above, as represented in the following Scheme: 
A further object of the invention are the novel compounds of general formulae (VII), (VIII), and (IX), in which R is defined as above; more particularly those of formulae (VIIa), (VIIIa), and (IXa), in which R is a hydrogen atom 
which compounds are useful as intermediates in the process shown in Scheme 1.
Analogously to 1,4,7,10-tetraazacyclododecane-1,4-diacetic acid, the novel chelating agents were prepared: xcex1,xcex1xe2x80x2-dimethyl-1,4,7,10-tetraazacyclododecane-1,4-diacetic acid, in which R in formula (I) is methyl; xcex1,xcex1xe2x80x2-diethyl-1,4,7,10-tetraazacyclododecane-1,4-diacetic acid, in which R is ethyl; xcex1,xcex1xe2x80x2-dibenzyl-1,4,7,10-tetraazacyclododecane-1,4-diacetic acid, in which R is benzyl.
In this case also the process for the preparation of the novel chelated complexes involves novel intermediate compounds, namely: xcex1-methyl-7-formyl-1,4,7,10-tetraazabicyclo[8.2.1]tridecane-13-on-4-acetic, xcex1-methyl-1,4,7,10-tetraazabicyclo[8.2.1]tridecane-13-on-4-acetic, xcex1,xcex1xe2x80x2-dimethyl-1,4,7,10-tetraazabicyclo[8.2.1]-tridecane-13-on-4,7-diacetic acids; 
xcex1-ethyl-7-formyl-1,4,7,10-tetraazabicyclo[8.2.1]tridecane-13-on-4-acetic, xcex1-ethyl-1,4,7,10-tetraazabicyclo-[8.2.1]tridecane-13-on-4-acetic, xcex1,xcex1xe2x80x2-diethyl-1,4,7,10-tetraazabicyclo[8.2.1]tridecane-13-on-4,7-diacetic acids; 
xcex1-benzyl-7-formyl-1,4,7,10-tetraazabicyclo[8.2.1]tridecane-13-on-4-acetic, xcex1-benzyl-1,4,7,10-tetraazabicyclo-[8.2.1]tridecane-13-on-4-acetic, xcex1,xcex1xe2x80x2-dibenzyl-1,4,7,10-tetraazabicyclo[8.2.1]tridecane-13-on-4,7-diacetic acids. 
It has also surprisingly been found that the hydrolysis of compounds (VII) and (VIII), and in particular (VIIa) and (VIIIa), in basic conditions (pH greater than 13), obtained by addition of a base, preferably NaOH or KOH, at high temperature (150-220xc2x0 C., preferably 180-200xc2x0 C.) and under pressure, yields compounds of formula (XI), in which R is defined as above. 
Said compounds are useful both as chelating agents for metal ions, and for the preparation of variously substituted chelating agents.
The process is particularly preferred for the preparation of the already known compound, 1,4,7,10-tetraazacyclododecane-1-acetic acid (see Meunier et al., Can. J. Chem., 73, 685, 1995), when R is a hydrogen atom. 
Also in this case the process for the preparation of this compound is more efficient and more suited for the industrial application than that already described in literature.
It is also an object of the invention the process for the preparation of compounds (I), comprising the steps represented in the following Scheme 3: 
in which step d) of the above Scheme is omitted and compound (V) is directly alkylated according to the procedure described in step e) of Scheme 1, to give compound (VII).
Particularly preferred is the process according to Scheme 3 for the preparation of compound (X), in which R is a hydrogen atom.
The compounds of general formula (I) are in their turn useful substrates for the preparation of compounds of general formula (XII), 
in which
R has the same meanings as defined above;
R1 is a hydrogen atom, a straight or branched or cyclic C1-C6 alkyl group, unsubstituted or substituted by 1 to 10 oxygen atoms, or a C1-C20 alkyl group, optionally interrupted by a phenylene, phenyleneoxy or phenylenedioxy in its turn substituted by a straight or branched C1-C6 alkyl group, unsubstituted or substituted by 1 to 7 hydroxy groups or 1 to 3 C1-C7 groups; the aromatic group being unsubstituted or substituted by alkoxy groups or by halogens, carboxy, carbamoyl, alkoxycarbonyl, sulfamoyl, hydroxyalkyl, amino, acylamino, acyl, hydroxyacyl;
Z is one of the following groups 
Rxe2x80x2 independently of R1, has the same meanings as R1 except for the hydrogen atom.
The compounds of formula (XII) are useful as chelating agents of paramagnetic metal ions, for the preparation of contrast agents for NMR diagnostics, as described for example in EP 325762.
Particularly preferred are the compounds of general formula (XIII), 
in which R and R1 have the meanings defined above.
It is also an object of the present invention the process for the preparation of compounds (XIII), starting from compounds (I) by alkylation, according to known methods, with an excess of an alkylating agent R1xe2x80x94CH(X1)xe2x80x94COY of formula (XIV), represented in the following Scheme 4: 
in which
R1 and R have the meanings as defined above;
X1 is a halogen or a sulfonic acid reactive residue;
Y is a xe2x80x94OH or xe2x80x94OR2 group, wherein R2 is a straight or branched C1-C4 alkyl group; when Y is xe2x80x94OR2, the ester groups are hydrolysed to obtain compounds of formula (XIII).
Alkylating agents of formula (XIV) corresponding to compound R1xe2x80x94CH(X1)xe2x80x94COOH, in which X1 is bromine or chlorine, are preferred, particularly preferred being the alkylating agents of formula X1CH2COOH, in which R is the hydrogen atom, and X1 is bromine or chlorine.
In the other cases the alkylating agent of formula (XIV) can be selected from the compounds which are already commercially available or whose preparation has already been described in literature (see for example WO 93/24469 or EP 325762), or those still to synthesize, using for example known methods for the preparation of suitable precursors (for example, for acyl chloride xcex1-halogen derivatives, see: Harpp et al., J. Org. Chem., 40. 3420. 1975), and subsequent transformation into the desired product.
Preferably R1 can be selected from the group consisting of: H or a straight or branched alkyl group, such as a methyl, ethyl, propyl, isopropyl, butyl, isobutyl group, in its turn substituted by hydroxy groups or interrupted by oxygen atoms, as defined above.
When an aromatic group is present in R1, particularly preferred are the phenyl, benzyl, phenylmethoxymethyl groups.
Particularly preferred are 3-(phenylmethoxy)-propanoic acid reactive derivatives, such as 2-bromo-3-(phenylmethoxy)propanoic acid, the preparation of which is described in Grossman et al., Chem. Ber., 91, 538, 1958, and 2-chloro-3-(phenylmethoxy)propanoic acid (CAS RN 124628-32-6), prepared analogously to the brominated derivative.
On the other hand, R2 is preferably selected from the group consisting of: methyl, ethyl, isopropyl, butyl, tert-butyl.
The reactive group X1 can be selected, by way of example, as already mentioned, from the group consisting of halogens (Cl, Br, I), or it can be a mesylate (MeSO2Oxe2x88x92), benzenesulfonyloxy (PhSO2Oxe2x88x92), nitrobenzenesulfonyloxy (p-NO2PhSO2Oxe2x88x92), tosylate (TsOxe2x88x92), or triflate (CF3SO3xe2x88x92) group.
The alkylation of compounds (I), when Y is the hydroxy group, can conveniently be performed as shown in Scheme 1 above.
Particularly preferred are the alkylating agents of general formula (XIV), in which Y is a hydroxy group, corresponding to bromoacetic acid (commercially available product), 2-bromopropionic acid (commercially available product), 2-bromobutyric acid (commercially available product).
The reaction solvent can suitably be selected from dipolar aprotic solvents, in particular from dimethylformamide (DMF), dimethylacetamide (DMA), dimethylsulfoxide (DMSO), acetonitrile (CH3CN) and N-methylpyrrolidone, and the reaction is carried out in the presence of an organic base, preferably a tertiary aliphatic amine selected from triethylamine (TEA), diisopropylethylamine and tributylamine.
In this case it can be convenient to also transform the acid groups present in the compound of formula (I), into the ester group xe2x80x94OR2, in order to promote the alkylation reaction, depending on the reactivity of the alkylating agent itself.
The reaction temperature will range, in this case, from 0 to 80xc2x0 C., depending on the reactivity of the selected alkylating agent.
In this case, the alkylation reaction will be followed by basic hydrolysis of the resulting diester, in conventional conditions, to obtain the desired compound of general formula (XIII).
By way of example of the huge potentialities provided by this synthetic route, the Experimental section describes the synthesis of the novel compound, xcex1,xcex1xe2x80x2-bis(methyl)-1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid: 
as well as that of xcex1,xcex1xe2x80x2-bis[(phenylmethoxy)methyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid 
which by catalytic hydrogenation, as described in example 6 of EP 325762, yields xcex1,xcex1xe2x80x2-bis(hydroxymethyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid. 
On the other hand, when in compounds (XII) Z is a phosphonic or phosphinic group, said compounds are prepared according to methods known in literature. More specifically, phosphonic derivatives are obtained from hydrochlorides or hydrobromides of the corresponding cyclic amines by reaction with formaldehyde and phosphorous acid (H3PO3) (see Sherry et al., Inorg. Chem., 28, 3336, 1989). The phosphine compounds are obtained from compound (I), previous esterification (e.g. formation of the t-butyl ester) by reaction with the corresponding phosphine derivative (such as diethoxymethylphosphine, (EtO)2PMe) and paraformal-dehyde, in anhydrous solvents. The resulting diester from the condensation is hydrolysed in acid solution at high temperature, to obtain the corresponding alkylphosphinic acid (see, e.g. Parker et al., J. Chem.Soc. Chem. Commun., 1738, 1990).
The synthesis of the novel compound, 1,4,7,10-tetraazacyclododecane-1,4-diacetic-7,10-dimethylenediphosphonic acid of formula (XV), starting from compound (I), according to methods known in literature, is reported in the Experimental section. 